Limits...
Distinct transduction profiles in the CNS via three injection routes of AAV9 and the application to generation of a neurodegenerative mouse model.

Huda F, Konno A, Matsuzaki Y, Goenawan H, Miyake K, Shimada T, Hirai H - Mol Ther Methods Clin Dev (2014)

Bottom Line: Using single-stranded adeno-associated virus serotype 9 (ssAAV9) vectors containing the neuron-specific synapsin-I promoter, we examined whether different administration routes (direct cerebellar cortical (DC), intrathecal (IT) and intravenous (IV) injections) could elicit specific transduction profiles in the CNS.In the cerebellar cortex, the DC and IT injection routes transduced all neuron types, whereas the IV injection route primarily transduced Purkinje cells.Thus, ssAAV9-mediated transduction areas, levels, and cell types change depending on the route of injection.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurophysiology, Gunma University Graduate School of Medicine , Maebashi, Japan ; Department of Physiology, Faculty of Medicine Universitas Padjadjaran , Bandung, Indonesia.

ABSTRACT
Using single-stranded adeno-associated virus serotype 9 (ssAAV9) vectors containing the neuron-specific synapsin-I promoter, we examined whether different administration routes (direct cerebellar cortical (DC), intrathecal (IT) and intravenous (IV) injections) could elicit specific transduction profiles in the CNS. The DC injection route robustly and exclusively transduced the whole cerebellum, whereas the IT injection route primarily transduced the cerebellar lobules 9 and 10 close to the injection site and the spinal cord. An IV injection in neonatal mice weakly and homogenously transduced broad CNS areas. In the cerebellar cortex, the DC and IT injection routes transduced all neuron types, whereas the IV injection route primarily transduced Purkinje cells. To verify the usefulness of this method, we generated a mouse model of spinocerebellar ataxia type 1 (SCA1). Mice that received a DC injection of the ssAAV9 vector expressing mutant ATXN1, a protein responsible for SCA1, showed the intranuclear aggregation of mutant ATXN1 in Purkinje cells, significant atrophy of the Purkinje cell dendrites and progressive motor deficits, which are characteristics of SCA1. Thus, ssAAV9-mediated transduction areas, levels, and cell types change depending on the route of injection. Moreover, this approach can be used for the generation of different mouse models of CNS/neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus

The ssAAV9 construct and a diagram showing the injection points. (a) Single-stranded adeno-associated virus serotype-9 (ssAAV9) DNA construct with its specific inverted terminal repeat (ITR) ends. We produced ssAAV9 vectors expressing the GFP under the control of the synapsin I promoter (Syn1) with the minimal CMV promoter at the 3′ end. Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was placed downstream of the GFP gene. (b) Positions of the ssAAV9 vector injections relative to the mouse head. ssAAV9 vectors were injected directly into the cerebellar cortex (direct cortical (DC) injection), intrathecally into the cisterna magna (intrathecal (IT) injection) or into the superficial temporal vein (intravenous (IV) injection). (c) Illustration of a sagittal section of the cerebellum and brainstem showing the positions of the DC and IT injections. For DC injections, ssAAV9 vectors were injected into lobule 6. For IT injections, the vectors were administered into the cisterna magna. (d) The injection point for the IV route through the superficial temporal vein, which runs adjacent to the mouse eye. DCN, deep cerebellar nuclei; IO, inferior olivary nucleus; PN, pontine nuclei; VN, vestibular nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362361&req=5

fig1: The ssAAV9 construct and a diagram showing the injection points. (a) Single-stranded adeno-associated virus serotype-9 (ssAAV9) DNA construct with its specific inverted terminal repeat (ITR) ends. We produced ssAAV9 vectors expressing the GFP under the control of the synapsin I promoter (Syn1) with the minimal CMV promoter at the 3′ end. Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was placed downstream of the GFP gene. (b) Positions of the ssAAV9 vector injections relative to the mouse head. ssAAV9 vectors were injected directly into the cerebellar cortex (direct cortical (DC) injection), intrathecally into the cisterna magna (intrathecal (IT) injection) or into the superficial temporal vein (intravenous (IV) injection). (c) Illustration of a sagittal section of the cerebellum and brainstem showing the positions of the DC and IT injections. For DC injections, ssAAV9 vectors were injected into lobule 6. For IT injections, the vectors were administered into the cisterna magna. (d) The injection point for the IV route through the superficial temporal vein, which runs adjacent to the mouse eye. DCN, deep cerebellar nuclei; IO, inferior olivary nucleus; PN, pontine nuclei; VN, vestibular nuclei.

Mentions: The ssAAV9 vectors expressing green fluorescent protein (GFP) were used to deliver a transgene into neurons in the brain. We used the enhanced neuron-specific synapsin I promoter with the minimal CMV promoter at the 3′ end as a promoter for transgene expression (Figure 1a), which was recently developed in our laboratory. Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was placed in the main AAV plasmid downstream of the GFP gene, which helped to enhance the stabilization of the mRNA and eventually increased the expression levels of the transgene.15 The ssAAV9 vectors were injected into the cerebellar cortex (direct cortical (DC) injection), the intrathecal space (IT injection) or the superficial temporal vein (IV injection) as illustrated in Figure 1b–d. We used 4-week-old mice for DC and IT injections and postnatal day 1 (P1) pups for IV injection because the transduction efficiency in the brain after IV injection decreases rapidly as mice mature.12


Distinct transduction profiles in the CNS via three injection routes of AAV9 and the application to generation of a neurodegenerative mouse model.

Huda F, Konno A, Matsuzaki Y, Goenawan H, Miyake K, Shimada T, Hirai H - Mol Ther Methods Clin Dev (2014)

The ssAAV9 construct and a diagram showing the injection points. (a) Single-stranded adeno-associated virus serotype-9 (ssAAV9) DNA construct with its specific inverted terminal repeat (ITR) ends. We produced ssAAV9 vectors expressing the GFP under the control of the synapsin I promoter (Syn1) with the minimal CMV promoter at the 3′ end. Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was placed downstream of the GFP gene. (b) Positions of the ssAAV9 vector injections relative to the mouse head. ssAAV9 vectors were injected directly into the cerebellar cortex (direct cortical (DC) injection), intrathecally into the cisterna magna (intrathecal (IT) injection) or into the superficial temporal vein (intravenous (IV) injection). (c) Illustration of a sagittal section of the cerebellum and brainstem showing the positions of the DC and IT injections. For DC injections, ssAAV9 vectors were injected into lobule 6. For IT injections, the vectors were administered into the cisterna magna. (d) The injection point for the IV route through the superficial temporal vein, which runs adjacent to the mouse eye. DCN, deep cerebellar nuclei; IO, inferior olivary nucleus; PN, pontine nuclei; VN, vestibular nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362361&req=5

fig1: The ssAAV9 construct and a diagram showing the injection points. (a) Single-stranded adeno-associated virus serotype-9 (ssAAV9) DNA construct with its specific inverted terminal repeat (ITR) ends. We produced ssAAV9 vectors expressing the GFP under the control of the synapsin I promoter (Syn1) with the minimal CMV promoter at the 3′ end. Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was placed downstream of the GFP gene. (b) Positions of the ssAAV9 vector injections relative to the mouse head. ssAAV9 vectors were injected directly into the cerebellar cortex (direct cortical (DC) injection), intrathecally into the cisterna magna (intrathecal (IT) injection) or into the superficial temporal vein (intravenous (IV) injection). (c) Illustration of a sagittal section of the cerebellum and brainstem showing the positions of the DC and IT injections. For DC injections, ssAAV9 vectors were injected into lobule 6. For IT injections, the vectors were administered into the cisterna magna. (d) The injection point for the IV route through the superficial temporal vein, which runs adjacent to the mouse eye. DCN, deep cerebellar nuclei; IO, inferior olivary nucleus; PN, pontine nuclei; VN, vestibular nuclei.
Mentions: The ssAAV9 vectors expressing green fluorescent protein (GFP) were used to deliver a transgene into neurons in the brain. We used the enhanced neuron-specific synapsin I promoter with the minimal CMV promoter at the 3′ end as a promoter for transgene expression (Figure 1a), which was recently developed in our laboratory. Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was placed in the main AAV plasmid downstream of the GFP gene, which helped to enhance the stabilization of the mRNA and eventually increased the expression levels of the transgene.15 The ssAAV9 vectors were injected into the cerebellar cortex (direct cortical (DC) injection), the intrathecal space (IT injection) or the superficial temporal vein (IV injection) as illustrated in Figure 1b–d. We used 4-week-old mice for DC and IT injections and postnatal day 1 (P1) pups for IV injection because the transduction efficiency in the brain after IV injection decreases rapidly as mice mature.12

Bottom Line: Using single-stranded adeno-associated virus serotype 9 (ssAAV9) vectors containing the neuron-specific synapsin-I promoter, we examined whether different administration routes (direct cerebellar cortical (DC), intrathecal (IT) and intravenous (IV) injections) could elicit specific transduction profiles in the CNS.In the cerebellar cortex, the DC and IT injection routes transduced all neuron types, whereas the IV injection route primarily transduced Purkinje cells.Thus, ssAAV9-mediated transduction areas, levels, and cell types change depending on the route of injection.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurophysiology, Gunma University Graduate School of Medicine , Maebashi, Japan ; Department of Physiology, Faculty of Medicine Universitas Padjadjaran , Bandung, Indonesia.

ABSTRACT
Using single-stranded adeno-associated virus serotype 9 (ssAAV9) vectors containing the neuron-specific synapsin-I promoter, we examined whether different administration routes (direct cerebellar cortical (DC), intrathecal (IT) and intravenous (IV) injections) could elicit specific transduction profiles in the CNS. The DC injection route robustly and exclusively transduced the whole cerebellum, whereas the IT injection route primarily transduced the cerebellar lobules 9 and 10 close to the injection site and the spinal cord. An IV injection in neonatal mice weakly and homogenously transduced broad CNS areas. In the cerebellar cortex, the DC and IT injection routes transduced all neuron types, whereas the IV injection route primarily transduced Purkinje cells. To verify the usefulness of this method, we generated a mouse model of spinocerebellar ataxia type 1 (SCA1). Mice that received a DC injection of the ssAAV9 vector expressing mutant ATXN1, a protein responsible for SCA1, showed the intranuclear aggregation of mutant ATXN1 in Purkinje cells, significant atrophy of the Purkinje cell dendrites and progressive motor deficits, which are characteristics of SCA1. Thus, ssAAV9-mediated transduction areas, levels, and cell types change depending on the route of injection. Moreover, this approach can be used for the generation of different mouse models of CNS/neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus